Leuconostoc mesenteroides B-1355 Mutants Producing Alternansucrases Exhibiting Decreases in Apparent Molecular Mass

Abstract

Mutants of Leuconostoc mesenteroides B-1355 exhibiting decreases in the apparent molecular mass of alternansucrase on sodium dodecyl sulfate (SDS)-polyacrylamide gels stained for enzyme activity were isolated after mutagenizing strain R15 with N-methyl-N(prm1)-nitro-N-nitrosoguanidine. Strain R15 was a UV mutant of strain B-1355 which was enriched for production of alternansucrase. All strains produced principal and minor alternansucrase bands on SDS gels when cultures were subjected to SDS-polyacrylamide gel electrophoresis (SDS-PAGE). The patterns of the principal and minor activity bands on our SDS gels did not result from dextran-enzyme complexes, because mutants constitutive for synthesis of glucosyltransferases (GTFs) on sugars other than sucrose produced activity bands after growth in glucose medium that were the same as those produced after growth in sucrose medium. Dextransucrase, which had been inactivated by heating at 45(deg)C, was reactivated when subjected to SDS-PAGE, showing that our SDS-PAGE conditions were reversibly denaturing. Thermal denaturation at 45(deg)C did not involve a dispersal of GTFs into subunits. Densitometry measurements showed a roughly linear relationship between enzyme activity and band intensity over a loading range of 0.2 to 0.8 mU per sample well. We concluded that SDS-PAGE followed by activity staining was a reliable method for estimating numbers and ratios of GTFs produced by Leuconostoc sp. in media containing sucrose.